摘要
研究背景
目前研究表明恶性肿瘤的发生与细胞分化紊乱或分化阻滞密切相关,而肿瘤治疗不管是放疗、化疗,还是分子靶向治疗以及生物治疗,都是采取杀灭肿瘤细胞的治疗理念,这些治疗方法在杀灭肿瘤细胞的同时对正常细胞也有一定的杀伤作用。越来越多的研究表明,在体内外诱导分化剂的作用下,恶性肿瘤细胞的形态、生物学或生物化学方面的诸多标志均向正常细胞的方向演化分化,甚至完全转变成正常细胞或接近正常细胞,这种现象称肿瘤细胞的再分化(redifferentiation),因此诱导分化治疗将成为未来肿瘤治疗的一个新的发展方向。
霍奇金淋巴瘤(Hodgkin lymphoma, HL)是一种淋巴造血系统的恶性肿瘤,1832年由英国的Thomas Hodgkin最早描述,其中经典型霍奇金淋巴瘤(classic Hodgkin lymphoma, cHL)约占霍奇金淋巴瘤病例的95%。该疾病的典型形态特点是少数的肿瘤性H/RS (Hodgkin/Reed-sternberg)细胞(<1%)浸润在大量的炎性反应背景细胞中。170多年来,H/RS细胞的本质以及发生发展机制等问题一直困扰着医学界。
Kuppers等采用显微切割技术从组织切片上挑取单个H/RS细胞,通过单细胞PCR技术分析Ig基因重排证实绝大部分的H/RS细胞来自淋巴器官生发中心(germinal center, GC)的前凋亡B细胞,也称为“残疾的B淋巴细胞”,该细胞具有B细胞表型丢失和发育不全的早期浆细胞分化潜能等B细胞分化紊乱的特点。寻找H/RS细胞发生过程中导致分化阻滞的关键靶点,调节其“开关”,使H/RS细胞复现B细胞的特征,回到B细胞分化的正常方向,是本研究的关键。
有报道反义CD99表达质粒转染人B淋巴瘤细胞株BJAB和IM9后,形态和免疫表型与H/RS细胞极为相似,提示CD99表达下调是H/RS细胞形成关键分子事件。转录因子PRDM1,诱导B细胞向浆细胞分化的一个开关,是浆细胞分化所必需的至关重要的调控因子。目前研究认为在cHL中PRDM1是一个重要的抑癌基因,其功能性失活阻断了B细胞向成熟浆细胞的分化,可能是cHL致病重要原因。H/RS细胞具有早期浆细胞分化潜能,诱导H/RS细胞向末端B细胞方向再分化必须打开PRDM1这一浆细胞调节关卡。最近研究发现miRNA在B细胞分化和B细胞恶性肿瘤中发挥重要作用。Nie等通过荧光素酶报告实验证实在cHL细胞株L428中PRDM1是miR-9调控的直接靶标,miR-9可以负性调控PRDM1的表达。因此,miR-9可能是导致H/RS细胞分化阻滞的又一重要靶标。
本课题组前期构建了CD99基因稳定过表达的cHL细胞株L428细胞亚系,命名为“L428-CD99”,在此基础上本研究采用免疫细胞化学、实时荧光定量RT-PCR、Western blot、免疫荧光共聚焦、原位杂交、miRNA反义寡核苷酸瞬时转染、生物信息学分析等方法,在对L428-CD99细胞亚系进行系列鉴定后,观测上调CD99后L428细胞B细胞分化相关表型、细胞形态学和细胞生物学行为等改变,明确上调CD99的H/RS细胞能否复现B细胞表型、打开向浆细胞分化的开关基因PRDM1并诱导向末端B细胞方向再分化可能性及调控机制,验证miR-9在I-H/RS细胞的表达、对靶点PRDM1的调控关系及探讨诱导再分化可能性,为进一步阐述H/RS细胞的发生发展机制提供参考依据。
研究目的
本研究拟通过四部分进行：
一、CD99和PRDM1在cHL组织和细胞水平的表达
观测CD99和PRDM1在淋巴结反应性增生(reactive lymphoid hyperplasia,RH)、cHL组织及其细胞株、B及T细胞淋巴造血系统肿瘤细胞株中的表达差异。
二、CD99调控PRDM1的表达诱导H/RS细胞再分化
探究CD99和PRDM1的调控关系,明确上调CD99基因对cHL细胞株L428细胞诊断标记、B细胞免疫表型以及浆细胞表型的影响,揭示CD99基因在cHL发生及分化所扮演的角色。
三、miR-9在H/RS细胞中的表达及对靶点PRDM1的调控
定量和定位、转染沉默验证miR-9在L428细胞的表达及对靶点PRDM1的调控。
四、CD99和miR-9相互调控关系的初步研究
初步实验并借助生物信息学分析CD99和miR-9的相互调控网络,探究CD99、miR-9和PRDM1的调控关系。
研究内容与方法
一、CD99和PRDM1在cHL组织和细胞水平的表达
收集cHL和RH组织标本,通过CD99和PRDM1的免疫组化比较其在组织上的表达差异,并进一步通过Western blot和免疫细胞化学方法检测CD99和PRDM1在六株B细胞来源和三株T细胞来源淋巴瘤细胞株的表达。
二、CD99调控PRDMl的表达诱导H/RS细胞再分化
1.L428-CD99细胞亚系的鉴定
对前期构建的慢病毒质粒稳定过表达CD99基因的L428-CD99克隆株,经荧光显微镜观察载体绿色荧光蛋白表达,利用实时荧光定量RT-PCR、Western blot和免疫荧光共聚焦检测CD99 mRNA和蛋白表达。
2.CD99基因过表达对cHL细胞株L428形态特征和生物学特性的影响
利用MTT实验、HE染色和鬼笔环肽染色检测CD99基因过表达对L428细胞增殖能力、形态大小以及细胞骨架蛋白的影响。
3.CD99基因上调对L428细胞分化相关蛋白的影响
经Western blot和免疫荧光共聚焦检测CD99基因过表达前后PRDM1蛋白的表达；利用免疫细胞化学和流式细胞检测cHL诊断标记(CD30和CD15)、B细胞分化相关抗原(CD10、CD19、CD20、CD45、CD79α、BCL6、PAX5和MUM1)以及浆细胞标记(CD38和CD138)。
三、miR-9在H/RS细胞中的表达及对靶点PRDM1的调控
1.检测miR-9在L428细胞的表达
利用免疫磁珠分选人正常淋巴结CD19+B淋巴细胞作对照；实时荧光定量RT-PCR和原位杂交检测CD19+B淋巴细胞和包括L428细胞株在内的8株淋巴瘤细胞株miR-9的表达。
2.瞬时干扰miR-9对L428细胞PRDM1蛋白表达和增殖能力的影响
miR-9反义寡核苷酸瞬时转染L428细胞,荧光显微镜观察分析转染效率,实时荧光定量RT-PCR检测干扰效率；Western blot和免疫荧光共聚焦检测敲低miR-9后PRDM1蛋白的表达；MTT实验分析miR-9反义寡核苷酸对L428细胞增殖能力的影响。
四、CD99和miR-9相互调控关系的初步研究
采用实时荧光定量RT-PCR检测CD99基因过表达前后miR-9的表达；利用实时荧光定量RT-PCR和Western blot检测miR-9干扰前后CD99的表达；通过生物信息学分析CD99和miR-9之间的信号调控通路。
结果
一、CD99和PRDM1在cHL组织和细胞水平的表达
1.在RH组织,CD99主要表达于淋巴滤泡外套区和滤泡间区,PRDM1在生发中心的明带呈散在弱阳性表达；62例cHL组织中,只有1例CD99在H/RS细胞表达阳性,所有病例PRDM1均呈阴性表达。
2.CD99和PRDM1蛋白在多发性浆细胞性骨髓瘤细胞株RPMI-8226中表达较高,在L428细胞和其他B细胞起源的淋巴瘤细胞株中表达较低甚至缺失。
二、CD99调控PRDM1的表达诱导H/RS细胞再分化
1.L428-CD99细胞亚系在荧光显微镜下均可见较强的绿色荧光,实时荧光定量RT-PCR、Western blot和共聚焦显微镜检测显示,与裸细胞组和空载体组比较,L428-CD99细胞亚系CD99基因和蛋白表达均增高。
2.MTT检测发现,CD99基因过表达组较空载体组增殖减慢,差异具有显著性(F=305.917,P=0.000)；HE染色后计数,L428-CD99细胞亚系20+uM细胞数占总细胞数比例为(50.187±2.518)%,较对照组细胞(77.588±5.580)%比例低,差异具有显著性(Z=-2.611,P=0.008)；鬼比环肽染色发现,与对照组相比,L428-CD99细胞丝足消失,皮层丝状肌动蛋白变薄。
3. Western blot和共聚焦检测结果显示,相对于裸细胞组和空载体组,CD99基因过表达组PRDMl蛋白表达明显增加；免疫细胞化学和流式细胞检测发现,CD99基因过表达后,与对照组相比,CD30、CD15和MUM1表达降低,CD10、CD19、CD79αBCL-6、PAX5和CD38表达增高,而CD20和CD138的表达没有明显改变。
三、miR-9在H/RS细胞的表达及对靶点PRDM1的调控
1.实时荧光定量RT-PCR检测发现,与分选CD19+B细胞和其他细胞株相比,miR-9在L428细胞的表达较高,差异均具有显著性(P<0.05)；原位杂交结果显示,miR-9的表达定位于胞浆,其在L428细胞呈弥漫强阳性表达,在DLBCL和Burkitt's淋巴瘤细胞株呈散在弱阳性表达,在T细胞来源的]KARPAS-299和Jurkat细胞表达阴性。
2.实时荧光定量RT-PCR检测发现,与对照组相比,miR-9反义寡核苷酸组miR-9的表达水平下降了50%左右,差异具有显著性(t=-5.208,P=0.035)。
3. Western blot和免疫荧光共聚焦结果显示,miR-9反义寡核苷酸组PRDM1蛋白表达相对裸细胞组和对照组显著升高。
4.MTT检测发现,裸细胞组、对照组(NCi)和miR-9反义寡核苷酸转染组(miR-9i)不同组别间细胞增殖活性差异不具有统计学意义(F=1.069,P=0.350)。
四、CD99和miR-9相互调控关系的初步研究
1.实时荧光定量RT-PCR结果显示,CD99转染L428细胞组相对于空载体组miR-9的表达水平显著降低,差异具有统计学意义(t=282.071,P=0.000)。
2.实时荧光定量RT-PCR检测发现,miR-9i组CD99的表达水平与对照组(NCi)和裸细胞组相当,差异不具有显著性(P=0.719和P=1.000)；Western blot结果显示,与对照组(NCi)和裸细胞组相比,miR-9反义寡核苷酸转染组CD99蛋白水平未发生明显改变。
3.生物信息学分析结果表明,CD99可能通过KPNB1的中间连接与SMAD3和SMAD4联系,进一步和MYC作用,调控miR-9的表达。
结论
1.CD99和PRDM1在cHL组织和细胞株呈较低或缺失表达。
2.L428-CD99细胞亚系过表达载体稳定、CD99基因和蛋白表达水平较高,细胞增殖减慢、体积变小、细胞骨架重构；CD99基因的过表达导致H/RS细胞丢失cHL诊断标记,重现B细胞表型,上调浆细胞分化关键因子PRDM1蛋白表达,出现浆细胞标记,具有前浆母细胞分化特征。
3.miR-9在L428细胞特异性高表达；miR-9可以负性调控PRDM1的表达。
4.CD99可以负向调控miR-9表达,但miR-9不能反作用于CD99; CD99很可能通过调控miR-9进而调节PRDM1的表达。
创新之处
1.提出H/RS细胞的产生和B细胞分化阻滞和分化紊乱密切相关,揭示了CD99、rniR-9和PRDM1与cHL的发生发展紧密关联；
2.初步揭示CD99基因可能通过调节miR-9介导的PRDM1表达,从而诱导H/RS细胞复现B细胞表型,并向末端B细胞方向再分化,为阐明cHL的发生机制提供帮助,为诱导分化治疗提供理论依据和精确靶标,具有原始创新性。
Backgroud
The present studies show that occurance of malignant tumors is closely related to the block or disorder of cell differentiation. The conventional therapies for treatment of malignant disorders, including radiotherapy, chemotherapy, molecular targeting and biological therapy are all base on the concept of eliminating tumor cells, of which will damage the nomal cells as well in the process. More and more studies indicate that induced by in vitro or in vivo agent, variant marks of the malignant tumor cells including those of the morphology, biology and biochemistry, evolve to normal differentiation and the tumor cells even transfer to complete or near complete normal cells. This phenomenon is termed tumor cell redifferentiation. Therefore, the inducing differentiation therapy may offer a new developing direction in the strategy of malignant tumor treatment.
Hodgkin lymphoma is the malignant tumor of lymphoid tissues firstly described by Dr. Thomas Hodgkin in 1832. The classic Hodgkin lymphoma (cHL) which is the major type of HL, accounts for about 95% of the cases. The cHL is characterized by the rare so-called Hodgkin/Reed-Sternberg (H/RS) tumour cells (< 1%) embedded in the background of predominant recative inflammatory cells. For more than 170 years, the nature and mechanism of development of H/RS cells remained illusive to be interpreted in medical circle.
Kuppers et al. picked out single H/RS cells from the biopsy specimen through microdissection technique, and single-cell PCR analysis of the immunoglobin (Ig) gene rearrangement of these cells proved that overwhelming majority of the H/RS originated from the pre-apoptotic germinal centre B cells called crippled B-cells in lymph organs. The H/RS cells are characterized by the loss of B-cell phenotype and evidence of abortive plasma cell differentiation. The key question in this study is to find out the pivotal targets that lead to differentiation block during generation of H/RS cells, and restore H/RS from B-cell makers through regulating these "switch" target back to the normal direction of B cell differentiation.
It has been reported that the morphology and immunophenotype of human B lymphoma cell lines (BJAB, IM9) transfected with antisense CD99 expression plasmid were similar with H/RS cells, suggesting that downregulation of CD99 expression is a key molecular event in the generation of H/RS cells. The transcription factor PRDM1, a "switch"of inducing B cells differentiation towards plasma cells, is a master regulator of plasma cell differentiation. The present studies suggest that PRDM1 is an important anti-oncogene in cHL, the functional inactivation of which may play a role in the pathogenesis of cHL by blocking the B cells differentiation towards plasma cells. H/RS cells have the potential of early plasma-cell differentiation, and induction H/RS cells towards terminal B cells must be based on increase of PRDM1 expression, a "switch" of plasma-cell differentiation. Recent studies have found that miRNAs play an important role in B cell differentiation and malignant B-cell lymphomas. Nie et al have confiremed that PRDM1 which is a direct target of miR-9, is negatively regulated by miR-9 in the cHL cell line of L428 by luciferase reporter experiments. Thus, miR-9 may be another important target for differentiated arrest of H/RS cells.
Our research group have previously constructed subseries of L428 cell line with stable overexpression of CD99 gene, named "L428-CD99". On this basis, we plan to identify the sub-cell line of L428-CD99, and detect the effect of upregulation of CD99 on cell morphology, the biological characteristics and differentiation-related cell phenotype in L428 cells, and explore whether overexpression of CD99 can be restored from B-cell phenotype, and induce redifferentiation of H/RS cells by upregulation of PRDM1, and verify expression of miR-9 in H/RS cells and its regulation on target PRDM1, and explore the possibility of induction of redifferentiation of H/RS cells, and provide reference for further elaborating mechanisms of generation and development of H/RS cells.
Objective
This study will be carried out in four parts:
1. The expression level of CD99 and PRDM1 in cHL tissues and cell lines
Detecting differences of CD99 and PRDM1 expression in reactive tissues of lymph node hyperplasia (RH), cHL tissues and cell lines, B-cell original and T-cell original lymphoma cell lines.
2. CD99 regulation on PRDM1 inducing redifferentiation of H/RS cells
Exploring the interaction between CD99 and PRDM1, and detecting the effect of upregulation of CD99 gene on diagnosis markers of cHL, B-cell differentiation-related phenotype and plasma-cell differentiation markers in L428 cells, revealing the role of CD99 gene in B cell differentiation and H/RS cell generation and transformation.
3. The expression of miR-9 in H/RS cells and its regulation on target PRDM1
Detecting miR-9 expression in cHL cell line quantitatively and positionally, and exploring the regulation of miR-9 on target PRDM1 by transient knockdown of miR-9 expression.
4. Preliminary investigations of regulation between CD99 and miR-9
Preliminary experiments and bioinformatics analysis of regulatory nets between miR-9 and PRDM1, so as to explore regulatory relation among CD99, miR-9 and PRDM1.
Contents and methods
1. CD99 and PRDM1 expression in cHL tissues and cell lines
The specimens of cHL cases and reactive lymph node hyperplasia were collected and analyzed by immunohistochemistry of CD99 and PRDM1. CD99 and PRDM1 protein expression were further measured by Western blot and immunocytochemistry in six B-cell origin and three T-cell origin lymphoma cell lines.
2. CD99 regulation on PRDM1 inducing redifferentiation of H/RS cells
(1) Identification of L428-CD99 sub-cell line
GFP expression was observed by fluorescence microscope in sub-cell line of L428-CD99 which was previously stablely transfeced by CD99 gene lentivirus vector. The mRNA and protein expression of CD99 was measured by real-time PCR, Western blot and confocal microscope.
(2) The effect of CD99 overexpression on morphology and biological characteristics of cHL cell line L428.
The effect of CD99 overexpression on proliferation, celluar size and cytoskeletal proteins was detected by MTT, HE staining and phalloidin staining.
(3) The effect of CD99 overexpression on differentiation-related proteins in L428 cells
The PRDM1 protein expression was detected by Western blot and confocal microscope after stable tansfection of CD99 gene. The diognosis markers of cHL (CD30, CD15), B cell differentiation-related proteins (CD10, CD19, CD20, CD45, CD79a, BCL6, PAX5 and MUM1) and plasma cell markers (CD38 and CD138) were detected by immunocytochemistry and flow cytometry.
3. The expression of miR-9 in H/RS cells and its regulation on target PRDM1
(1) Detection of miR-9 expression in L428 cells
Magnetic separation of CD19+ B lymphocytes was performed from normal lymph nodes as a control. The miR-9 expression was detected by real-time PCR and in situ hybridization (ISH) in isolated CD19+ B-cell subsets and eight lymphoma cell lines including L428 cells.
(2) The effect of transient interference of miR-9 on PRDM1 protein expression and cellular proliferation in L428 cells
Transfection efficiency was analyzed by fluorescence microscope, and interference efficiency was detected by real-time PCR after transient transfection of miR-9 antisense oligonucleotide. The PRDM1 protein expression was detected by Western blot and confocal microscope after knockdown of miR-9. The effect of miR-9 antisense oligonucleotide on proliferation of L428 cells was detected by MTT assay.
4. Preliminary investigations of regulation between CD99 and miR-9
The expression of miR-9 was detected by real-time PCR after upregulation of CD99 gene. The expression of CD99 was detected by real-time PCR and Western blot after interference of miR-9. Bioinformatics analysis of signal regulatory pathway between miR-9 and PRDM1 was performed.
Results
1. CD99 and PRDM1 expression in cHL tissues and cell lines
(1) CD99 was mainly expressed in mantle zone B cells and interfollicular area of reactive lymphoid hyperplasia tissues (RH) and PRDM1 was expressed in light zones of germinal centers (GC). The positivity for CD99 was found in 1 of the 62 cHL cases examined, and PRDM1 expression was not found in H/RS cells of all cHL cases.
(2) CD99 and PRDM1 were highly expressed in multiple myeloma (MM) cell line RPMI-8226, and lowly or absently expressed in L428 cell and other B-cell origin of lymphoma cell lines.
2. CD99 regulation on PRDM1 inducing redifferentiation of H/RS cells
(1) The strong green fluorescence was observed in L428-CD99 sub-cell line under fluorescence microscope. As indicated, CD99-upregulated L428 cells showed increased expression of CD99 gene and protein expression compared with mock and empty vecter groups using real-time PCR, Western blot and confocal microscopy analysis.
(2) MTT assay indicated that CD99-overexpressed L428 cells showed reduced growth compared with controls, with significant difference (F= 305.917, P= 0.000). H&E staining analysis showed that the ratio of 20+ uM cells occupying the total cell number in L428-CD99 cells is (50.187±2.518)%, compared with that of (77.588±5.580)% in control groups, with significant difference (Z=- 2.611, P= 0.008). When cells were stained with phalloidin-FITC to label filamentous actin, we clearly observed the disappearance of filopodia and thinningz of cortical filamentous actin in CD99-transfected L428 cells compared with control cells.
(3) Western blot and confocal microscopy analysis showed that L428 cells with overexpression of CD99 showed a concomitant increasing expression of PRDM1 protein compared with mock and empty vecter groups. The L428 cells transfected with CD99 gene showed the decrease of CD30, CD 15 and MUM1 expression, and increase of CD10, CD19, CD79a, BCL-6, PAX5 and CD38 expression. There was no significant change for CD20 and CD138 by immunocytochemistry and flow cytometry analysis, compared with those of control cells.
3. The expression of miR-9 in H/RS cells and its regulation on target PRDM1
(1) The results of qRT-PCR showed that expression of miR-9 in L428 cells is markedly higher than those of isolated CD19+ B-cell subsets and other lymphoma cell lines, with significant difference (P< 0.05). In situ hybridization showed that miR-9 was extensively localized in the cytoplasm. A strong and uniform cytoplasmic signal was observed in cHL cell line, compared with scattered signal in DLBCL and Burkitt's lymphoma cell lines, while no signal was detected in T-cell origin of KARPAS-299 and Jurkat cells.
(2) The results of real-time PCR showed that enforced miR-9 inhibitor resulted in an almost 50% decrease of miR-9 expression in L428 cells (t=-5.208, P= 0.035).
(3) The results of Western blot showed that the L428 cells transfected with miR-9 inhibitor showed an increase of PRDM1 expression, compared with those of mock and control grpups.
(4) No remarkable differences in cell growth were observed among the mock, negative control and miR-9 inhibitor groups (F= 1.069, P= 0.350).
4. Preliminary investigations of regulation between CD99 and miR-9
(1) The results of real-time PCR showed that expression of miR-9 was significantly downregulated by introduction of CD99 into L428 cells (t= 282.071, P= 0.000).
(2) The CD99 expression remained generally unaffected in cells transfected with miR-9 inhibitor, compared with those of mock and negative control groups by real-time PCR (P= 0.719, P= 1.000). The same result was also confirmed by western blot.
(3) Bioinformatics analysis showed that CD99 may regulate miR-9 expression, through KPNB1-(SMAD3, SMAD4)-MYC signal pathway.
Conclusion
1. Both of CD99 and PRDM1 were lowly expressed or absent in cHL tissues and cell lines.
2. The overexpressed CD99 gene lentivirus vector in L428-CD99 sub-cell line is stable. The overexpressed CD99 gene leads to upregulation of CD99 mRNA and protein expression, reduced cell growth, decrease of cellular size and reorganization of actin cytoskeleton in L428 cells. Furthermore, upregulation of CD99 leads to a decrease in cHL diagnosis marker CD30 and CD 15, restoration of B-cell makers, and increase in plasma cell differentiation key factor PRDM1 protein along with plasma cell marker CD38, suggesting characteristics of pre-plasmablast cells.
3. MiR-9 is specificly overexpressed in L428 cells and may negatively regulate the expression of PRDM1.
4. CD99 negatively regulate miR-9, while miR-9 can not react up on CD99 expression. CD99 may regulate PRDM1 expression through controlling miR-9 expression.
Discoveries and innovations
1. Our results suggest that generation of H/RS is closely associated with B-cell differentiation block and disorders, reveal that CD99, miR-9 and PRDM1 is tightly related to generation and development of cHL.
2. We have preliminarily revealed that CD99 may regulate miR-9 to mediate PRDM1 expression, which induces restoration of B-cell makers and differentiation towards terminal B cells. This research provids the theoretical basis and accurate target for differentiation therapy, with original creativity.
引文
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